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molecules
Article
Implementation of an Analytical Method for
Spectrophotometric Evaluation of Total Phenolic Content in
Essential Oils
Delia Michiu 1 , Maria-Ioana Socaciu 1 , Melinda Fogarasi 1 , Anamaria Mirela Jimborean 1, *, Floricuţa Ranga 2 ,
Vlad Mureşan 1 and Cristina Anamaria Semeniuc 1, *
1 Department of Food Engineering, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
3-5 Mănăştur Str., 400372 Cluj-Napoca, Romania; delia.michiu@usamvcluj.ro (D.M.);
maria-ioana.socaciu@usamvcluj.ro (M.-I.S.); melinda.fogarasi@usamvcluj.ro (M.F.);
vlad.muresan@usamvcluj.ro (V.M.)
2 Department of Food Science, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
3-5 Mănăştur Str., 400372 Cluj-Napoca, Romania; floricutza_ro@yahoo.com
* Correspondence: mirela.jimborean@usamvcluj.ro (A.M.J.); cristina.semeniuc@usamvcluj.ro (C.A.S.);
Tel.: +40-264-596-384 (A.M.J. & C.A.S.)
Abstract: Over the past decade, there has been growing interest in polyphenols’ research since these
compounds, as antioxidants, have several health benefits, such as preventing neurodegenerative
diseases, inflammation, cancer, cardiovascular diseases, and type 2 diabetes. This study implements
an analytical method to assess the total phenolic content (TPC) in essential oils using Folin–Ciocalteu’s
phenol reagent and quantifies the individual phenolic compounds by liquid chromatography. Thus,
the research design and methodology included: (1) extraction of essential oil from dried thyme
leaves by hydrodistillation; (2) spectrophotometric measurement of TPC by Folin–Ciocalteu method;
Citation: Michiu, D.; Socaciu, M.-I.; and (3) identification and quantification of individual phenolic compounds by high-performance
Fogarasi, M.; Jimborean, A.M.; Ranga, liquid chromatography-diode array detection/electrospray ionization mass spectrometry (HPLC-
F.; Mureşan, V.; Semeniuc, C.A. DAD-ESI-MS). Results revealed a TPC of 22.62 ± 0.482 mg GAE/100 µL and a polyphenolic profile
Implementation of an Analytical characterized by phenolic acids (52.1%), flavonoids (16.1%), and other polyphenols (31.8%). Thymol,
Method for Spectrophotometric salvianolic acid A, and rosmarinic acid were the major compounds of thyme essential oil. The
Evaluation of Total Phenolic Content
proposed analytical procedure has an acceptable level of repeatability, reproducibility, linearity, LOD
in Essential Oils. Molecules 2022, 27,
(limit of detection), and LOQ (limit of quantification).
1345. https://doi.org/10.3390/
molecules27041345
Keywords: total phenolic content; essential oils; Folin–Ciocalteu method; UV-Vis spectrophotometry;
Academic Editor: Francesco Cacciola polyphenolic compounds; HPLC-DAD-ESI-MS analysis
Received: 29 January 2022
Accepted: 14 February 2022
Published: 16 February 2022
1. Introduction
Publisher’s Note: MDPI stays neutral
Polyphenols are compounds found naturally in plants, as they are synthesized in
with regard to jurisdictional claims in
published maps and institutional affil-
their tissues, in fruits and vegetables [1]. Endogenous synthesis of phenolic compounds
iations.
is due to plants’ response to ecological and physiological pressures such as pathogen and
insect attacks, UV radiation, and wounding [2]. These compounds are recognized for their
antioxidant properties, and their potential benefits have been largely studied [3–5]. The
antioxidant activity of polyphenolic compounds is related to their reactive radical species
Copyright: © 2022 by the authors. inactivation capability; neutralization occurs when an antioxidant transfers its electron
Licensee MDPI, Basel, Switzerland. and/or hydrogen atom to the radical [6]. Previous studies have shown that many dietary
This article is an open access article polyphenolic constituents derived from plants are more effective antioxidants in vitro
distributed under the terms and than vitamins E or C, and thus might contribute significantly to the protective effects
conditions of the Creative Commons in vivo [7]. Recent evidence has proposed that a high dietary intake of polyphenols may
Attribution (CC BY) license (https:// be inversely associated with overall and cardiovascular disease-related mortality, certain
creativecommons.org/licenses/by/ cancers, anthropometric measurements, and mood disorders [8].
4.0/).
Molecules 2022, 27, 1345. https://doi.org/10.3390/molecules27041345 https://www.mdpi.com/journal/moleculesMolecules 2022, 27, 1345 2 of 13
Polyphenolic compounds are phytochemicals consisting of flavonoids and nonflavonoids,
subdivided into multiple subclasses depending on the number of phenol units within their
molecular structure, substituent groups, and/or the linkage type between phenol units [9].
Flavonoids are characterized by the presence of a 15-carbon (C6–C3–C6) backbone consist-
ing of two phenyl units (A and B) and a heterocyclic unit (C) [10]. Structurally, they are
grouped into subclasses, such as 2-phenylchromans (flavonoids, including flavanones,
flavones, flavonols, flavanols, flavanonols, anthocyanins, and anthocyanidins) and 3-
phenylchromans (isoflavonoids, including isoflavones, isoflavans, and pterocarpans) [9,11].
Non-flavonoid phenolic constituents include phenolic acids (hydroxycinnamic and hy-
droxybenzoic acids), volatile phenols, stilbenes (monomeric and oligomeric stilbenes),
coumarins (simple coumarins, furanocoumarins, pyranocoumarins, and pyron-ring sub-
stituted coumarins), lignins (guaiacyl and guaiacyl-syringyl), lignans (furofuran, furan,
dibenzylbutane, dibenzylbutyrolactone, aryltetralin, arylnaphthalene, dibenzocyclooctadi-
ene, and dibenzylbutyrolactol), hydrolysable tannins (gallotannines, ellagitannines, and
complex tannins), and condensed tannins (called procyanidins) [2,9,12–16].
Aromatic plants and their essential oils are rich in phenolic compounds, so-called
phenylpropanoids [17]. While the main constituents of essential oils are generally mono-
and sesquiterpenes, phenylpropanoid compounds are predominant in certain plant families.
In addition, some terpenes are phenol derivatives, namely phenolic terpenes [18,19]. More
recent evidence shows that the chemical composition of essential oils depends on several
factors such as the domestication of plants, environmental conditions of plants growing,
genetic differences, the plant part used for their isolation. Moreover, the harvesting season
of plants and the extraction method, generally affect extraction yield and composition of
essential oil [20,21].
The Folin–Ciocalteu method is the most used to assess total phenolic content (TPC) in
essential oils. Its principle consists of phenolic compounds oxidation from the analyzed
sample by the Folin–Ciocalteu reagent. This reagent is formed from a mixture of phospho-
tungstic acid (H3 PW12 O40 ) and phosphomolybdic acid (H3 PMo12 O40 ). After oxidation of
the phenols, it is reduced to a mix of blue oxides of tungsten (W8 O23 ) and molybdenum
(Mo8 O23 ). The blue coloration produced has maximum absorption in the region of 750 nm,
and it is proportional to the total quantity of phenolic compounds initially present [22]. Gal-
lic acid is usually used as the standard for quantitatively estimating phenolic compounds
in the sample. Results are expressed as mg of gallic acid equivalents (GAE) per volume
(mL or 100 µL) or mass (g) of essential oil.
There are a few studies in the literature on TPC measurement in thyme essential oil.
For example, Lin et al. (2009) have investigated TPC levels in commercially available
essential oils of Thymus vulgaris and Thymus serpyllum [23]. In addition, in 2014, Fatma et al.
have measured TPC in essential oils of dried Thymus hirtus sp. algeriensis collected from
different locations of Tunisia and in 2018, Aljabeili et al. in the essential oil of dried Thymus
vulgaris from Egypt [24,25]. Furthermore, Mancini et al., in 2015, have assessed TPCs in
essential oils of fresh Thymus vulgaris sampled from different areas of Italy [26]. More
recently, Mutlu-Ingok et al. (2021) have quantified total phenolics in a commercial sample
of thyme essential oil [27]. However, there is limited research on the utilization of liquid
chromatography in evaluating polyphenolic profiles of thyme essential oils [24].
None of the above-mentioned reports gives a detailed description of the protocol
followed to evaluate TPC in essential oil. In general, information about dilutions used,
blank sample preparation, or standard concentrations is missing. Therefore, this study was
designed as a detailed protocol for assessing TPC in essential oils, using the Folin–Ciocalteu
method, to be implemented in an analytical laboratory. The experimental design included
extraction of thyme essential oil by hydrodistillation, spectrophotometric evaluation of
TPC, and determination of phenolic compounds profile in thyme essential oil by an HPLC-
DAD-ESI-MS method. As far as we are aware, this is the first study demonstrating the
suitability of this method for its intended use.Molecules 2022, 27, 1345 3 of 13
2. Results and Discussion
The extraction yield of thyme essential oil (2.2%) was calculated as the volume of
essential oil (mL) per dried leaves’ weight (g) and multiplied by 100 [28].
Instrument calibration is an important stage in most measurement procedures. It
involves (1) preparing a set of standards containing a known amount of the analyte of
interest, the gallic acid in our case, (2) measuring the instrument response (absorbance
value) for each standard, and (3) establishing the relationship between the instrument
response and analyte concentration. This relationship is further used to transform measure-
ments made on test samples into estimates of the amount of analyte present, as shown in
Section 3.2.5. As calibration is a common and essential step in analytical methods, analysts
must understand how to set up calibration experiments and evaluate results [29].
For constructing the calibration curve, five gallic acid standards were prepared in
duplicate (in concentrations ranging from 0.25 to 1.25 mg/mL) and following the Folin-
Ciocalteu method. Data from the calibration experiment were then plotted: on the y-axis,
values of absorbances, and the x-axis, values of standards’ concentrations. Next, a linear
regression through the origin was performed by using the five following data points:
1 (0.25, 0.187), 2 (0.50, 0.392), 3 (0.75, 0.613), 4(1.0, 0.828), and 5 (1.25, 1.058), to establish
the equation c = 0.8323 × Abs.; R2 = 0.9991 that best describes the linear relationship
between absorbance value and gallic acid level, where c is the gallic acid concentration,
0.8323 is the slope of the regression line, and Abs. is the absorbance value at 750 nm. Finally,
a regression analysis was carried out using the regression tool in Excel’s “Data Analysis
ToolPak” to determine the correlation coefficient, r, and significance of the correlation,
p-value. The regression output created revealed several statistical parameters (see Table 1).
The correlation coefficient (r), described as “Multiple R” in Excel output, indicates the
strength and direction of the linear relationship between the two variables. It ranges from
−1 (negative correlation) to +1 (positive correlation). When the r-value is in-between
0.00–0.19, the correlation is very weak; weak between 0.20 and 0.39, moderate between
0.40 and 0.59, strong between 0.60 and 0.79, and very strong between 0.80 and 1.0. The
significance of the correlation is interpreted based on the p-value as follows: p ≥ 0.05NS ,
not significant trend; 0.01 ≤ p < 0.05, weakly significant; 0.005 ≤ p < 0.01, mildly significant;
0.001 ≤ p < 0.005, moderately significant; p < 0.001, strongly significant [30]. Considering
the values resulting for r (0.999) and p (2.64 × 10−12 ), a very strong positive correlation
was found between the two variables of the calibration curve, demonstrating its linearity
within the range of the tested standards.
Table 1. Statistics of the calibration curve.
Regression Statistics
Multiple R 0.999118
R Square 0.998238
Adjusted R Square 0.998017
Standard Error 0.014533
Observations 10
ANOVA
df SS MS F Significance F
Regression 1 0.957031 0.957031 4531.264 2.64 × 10−12
Residual 8 0.00169 0.000211
Total 9 0.958721
Standard
Coefficients t Stat p-value Lower 95% Upper 95% Lower 95% Upper 95%
Error
Intercept −0.03915 0.010778 −3.63243 0.006662 −0.064 −0.0143 −0.064 −0.0143
X Variable
0.875 0.012999 67.31466 2.64×10−12 0.845025 0.904975 0.845025 0.904975
1Molecules 2022, 27, 1345 4 of 13
Limit of detection (LOD) and limit of quantification (LOQ) are two fundamental
elements of method validation that define the limitations of an analytical method [31]. The
LOD is the lowest amount of the analyte that can be detected by the method at a specified
level of confidence. The LOQ is the lowest concentration of analyte that can be determined
with an acceptable level of uncertainty and can, therefore, be set arbitrarily as the required
lower end of the method working range [32].
To calculate the LOD (Equation (2)) and LOQ (Equation (3)) of this analytical procedure,
first, we estimated the standard deviation (SD) of intercept with the following Equation (1):
√
SD of intercept = 0.010778 × 5 (1)
where 0.010778 is the intercept’s standard error (SE) (see the Table above) and 5 is the
number of calibration standards.
0.0241
LOD = 3.3 × (2)
0.8323
0.0241
LOQ = 10 × (3)
0.8323
where 3.3 is the factor for LOD, 10 is the factor for LOQ, 0.0241 is the intercept’s standard
deviation (SD), and 0.8323 is the slope of the calibration curve.
A LOD of 0.096 mg/mL and a LOQ of 0.290 mg/mL thus resulted. Therefore, even
though the calibration curve is linear within the range of 0.25–1.25 mg GA/mL, phenolics
can be quantified using this protocol up from a concentration of 0.29 mg GAE/mL oil.
Folin-Ciocalteu is a common method for assessing TPC, and many studies have
reported this technique for phenolic compounds quantification in thyme essential oil.
However, some necessary information in reproducing this method is missing, such as the
sample dilution ratio. Since thyme essential oil has a content of phenolic compounds that
exceeds the upper limit of quantification (ULOQ), to evaluate TPC, a dilution of the sample
is needed. The upper limit of quantification (ULOQ) is the maximum analyte concentration
that can be quantified with acceptable precision and accuracy (bias) in a sample. In general,
the ULOQ is identical to the highest concentration of calibration curve [33]. Thus, when
a sample is too concentrated, this must be diluted before analysis so that the analyte
concentration falls in the middle of the calibration curve linear range [29]. Taking this into
account, in the current research, we prepared a series of 16 dilutions of thyme essential oil
in methanol (from 1:125 to 1:500) and subjected them to the Folin–Ciocalteu assay. Values of
absorbances read at 750 nm were recorded in the worksheet from Table 2. The concentration
of each dilution of thyme essential oil (x from the calibration curve), expressed in mg
GAE/mL, was calculated by dividing the absorbance value read (y value) to 0.8323 (a value,
the slope). As data are expressed as the mean value ± standard deviation (SD) of the three
replicates, these values, including those of variation coefficients (CVs), were calculated for
each dilution and recorded in the worksheet from Table 2. Generally, the CV, also known as
relative standard deviation (RSD), a measure of precision between replicate measurements,
should beMolecules 2022, 27, 1345 5 of 13
Table 2. Worksheet for recording values of absorbances corresponding essential oil samples.
Absorbance Value TPC
TEO Sample Dilution at 750 nm [mg GAE/100 µL]
Factor
Mean SD CV Mean SD CV
Dilution of 1:500 501 0.57 k 0.011 2.0 28.39 0.559 2.0
Dilution of 1:475 476 0.56 k 0.008 1.4 26.82 0.375 1.4
Dilution of 1:450 451 0.57 k 0.019 3.3 25.65 0.850 3.3
Dilution of 1:425 426 0.66 j 0.010 1.6 27.91 0.447 1.6
Dilution of 1:400 401 0.71 i 0.009 1.3 28.28 0.376 1.3
Dilution of 1:375 376 0.70 i 0.011 1.6 26.47 0.431 1.6
Dilution of 1:350 351 0.72 hi 0.008 1.2 25.30 0.292 1.2
Dilution of 1:325 326 0.76 h 0.005 0.7 24.73 0.163 0.7
Dilution of 1:300 301 0.75 h 0.016 2.1 22.62 0.482 2.1
Dilution of 1:275 276 0.84 g 0.003 0.4 23.12 0.083 0.4
Dilution of 1:250 251 0.92 f 0.023 2.5 22.98 0.575 2.5
Dilution of 1:225 226 1.04 e 0.032 3.1 23.57 0.729 3.1
Dilution of 1:200 201 1.18 d 0.017 1.4 23.74 0.342 1.4
Dilution of 1:175 176 1.23 c 0.021 1.7 21.63 0.362 1.7
Dilution of 1:150 151 1.63 b 0.014 0.8 24.63 0.209 0.8
Dilution of 1:125 126 3.41 a 0.0 0.0 42.90 0.0 0.0
TEO—thyme essential oil; SD—standard deviation; CV—coefficient of variation. Different letters in the same row
indicate a statistically significant difference at p < 0.05 (Tukey’s test).
Significantly lower levels were found by Lin et al. (2009) in Thymus vulgaris essential
oil (11.54 mg GAE/g) and Thymus serpyllum essential oil (55.10 mg GAE/g), by Fatma et al.
(2014) in essential oils of Thymus hirtus sp. algeriensis collected from different locations of
Tunisia (7.08–8.81 mg GAE/g), by Mancini et al. (2015) in essential oils of Thymus vulgaris
sampled from different areas of Italy (77.6–165.1 mg GAE/g), and by Mutlu-Ingok et al.
(2021) in the commercial sample of thyme essential oil (0.019 mg GAE/100 µL) [23,24,26,27].
However, comparable levels were reported by Aljabeili et al. (2018) in the Egyptian essential
oil of Thymus vulgaris (177.3 mg GAE/g) [25].
Table 3 shows individual polyphenolic compounds quantified in the thyme essential oil
taken in this study by HPLC-DAD-ESI-MS analysis. Eighteen compounds were identified
in this essential oil and grouped into three chemical classes (phenolic acids-PAs, flavonoids-
FVs, and other phenols-OPs) that included seven compound types (hydroxybenzoic acids-
HBA, hydroxycinnamic acids-HCAs, flavanols-FVols, flavones-FVes, phenolic terpenes-PTs,
caffeic acid derivatives-CADs, and coumaric acid derivatives-CODs). The most abundant
constituents were phenolic acids [52.1% (4.5% HBAs, 18.9% HCAs, 21.9% CADs, and
6.7% CODs), followed by other polyphenols [31.8%, represented by PTs], and flavonoids
[16.1% (2.0% FVols and 14.0% FVes)]. The major compounds in thyme essential oil were the
thymol (333.37 µg/mL), a phenolic terpene, salvianolic acid A (223.33 µg/mL), a caffeic
acid derivative, and rosmarinic acid (114.73 µg/mL), an hydroxycinnamic acid.
Although the volatile composition of thyme essential oil by gas-chromatography is well-
studied, there is a lack of studies on its polyphenolic composition using liquid chromatography.
Our literature review revealed two such studies, the one of Hajimehdipoor et al. (2010) on
essential oil of Thymus vulgaris L. [35], and the one of Fatma et al. (2014) on essential oil
of Thymus hirtus sp. algeriensis [24]. Thymol was also the majority compound in thyme
essential oil studied by Hajimehdipoor et al. (2010), followed by carvacrol [35]. However,
Fatma et al. (2014) reported a different polyphenolic profile of their thyme essential oil,
with hydroxybis, tyrosin, and hydroxyphenyl acetic acid as main compounds [24].Molecules 2022, 27, 1345 6 of 13
Table 3. Content of polyphenolic compounds (µg/mL) in thyme essential oil of the current study
and comparison with literature data.
Crt. Chemical Type of Literature Data
Compound TEO (µg/mL)
No. Class Compound Studied Thymus Genotypes Analytical Method/Content Ref.
1 Gallic acid PAs HBA 3.40 ± 0.211 Thymus hirtus sp. algeriensis HPLC-DAD/433.92 µg/g dw [24]
2 p-Hydroxybenzoic acid PAs HBA 65.14 ± 0.378 - - -
3 Caffeic acid PAs HCA 49.80 ± 0.313 - - -
4 Epicatechin FVs FVol 30.45 ± 0.313 – -
5 p-Coumaric acid PAs HCA 62.91 ± 0.375 - - -
6 Ferulic acid PAs HCA 58.01 ± 2.473 Thymus hirtus sp. algeriensis HPLC-DAD/433.92 µg/g dw [24]
7 Apigenin-7-O-glucoside FVs FVe 36.85 ± 0.976 - - -
8 Luteolin-7-O-glucoside FVs FVe 66.93 ± 4.036 - - -
9 Rosmarinic acid PAs HCA 114.73 ± 2.849 - - -
10 Naringenin FVs FVe 35.26 ± 1.889 - - -
11 Apigenin FVs FVe 47.29 ± 0.585 - - -
12 Luteolin FVs FVe 25.68 ± 1.335 - - -
13 Methyl rosmarinate PAs COD 101.91 ± 2.192 - - -
14 Rosmadial OPs PT 92.65 ± 2.313 - - -
15 Salvianolic acid C PAs CAD 107.83 ± 2.334 - - -
16 Salvianolic acid A PAs CAD 223.33 ± 21.451 - - -
17 Carvacrol OPs PT 55.03 ± 0.0 Thymus vulgaris L. HPLC-DAD/4.3% (w/w) [35]
18 Thymol OPs PT 333.37 ± 42.480 Thymus vulgaris L. HPLC-DAD/40.4% (w/w) [35]
Total content 1510.58 - - -
TEO—thyme essential oil; PAs—phenolic acids; FVs—flavonoids; OPs—other polyphenols; HBA—hydroxybenzoic acid;
HCA—hydroxycinnamic acid; FVol—flavanol; FVe—flavone; PT—phenolic terpene; CAD—caffeic acid deriva-
tive; COD—coumaric acid derivative; Ref.—bibliographic reference. Values are expressed as mean ± standard
deviation of three replicates.
3. Materials and Methods
3.1. Plant Material and Extraction of Thyme Essential Oil
For essential oil extraction, dried thyme leaves of Thymus vulgaris were purchased
from a company that markets food ingredients (Solina Group, Alba Iulia, Romania http:
//www.solina-group.ro; accessed on 24 January 2022). Thyme essential oil was obtained
by hydrodistillation using a Clevenger-type apparatus (S.C. Energo-Metr S.R.L., Odorheiu
Secuiesc, Romania); 50 g of thyme leaves were boiled, for 3 h, in 750 mL distilled water.
The collected essential oil was dried over sodium sulphate anhydrous and stored at 4 ◦ C
until analysis.
3.2. Spectrophotometric Evaluation of Total Phenolic Content (TPC) in Essential Oil by
Folin-Ciocalteu Method
The operating procedure proposed in this paper is a deliverable resulting from im-
plementing this method in our research laboratory [19]. Step-by-step instructions are
below detailed.
3.2.1. Scope
The present operating procedure specifies a method for assessing TPC in essential oil
using the Folin–Ciocalteu method. This method is suitable for essential oil having a TPC of
up to 42 mg gallic acid equivalent (GAE)/100 µL.
3.2.2. Principle
A test portion (100 µL) of diluted essential oil is dissolved in distilled water (6.0 mL).
Next, 2 N Folin–Ciocalteu’s phenol reagent (0.5 mL), 0.71 M sodium carbonate solution
(1.5 mL), and the rest of distilled water (1.9 mL) are added to reach a 10 mL volume. After
a fixed period of reaction (2 h), the content of phenols is calculated from a photometric
measurement of the blue mixture of tungsten (W8 O23 ) and molybdenum (Mo8 O23 ) oxides.
3.2.3. Preparation of Working and Standard Solutions
Only reagents of recognized analytical grade and distilled water were used.
Reagents:
• Gallic acid (2699.1, Carl Roth GmbH & Co. KG, Karlsruhe, Germany)
• Folin–Ciocalteu’s phenol reagent (F9252, Sigma-Aldrich Co., Saint Louis, MO, USA)Molecules 2022, 27, 1345 7 of 13
• Sodium carbonate anhydrous (27767.295, VWR, Leuven, Belgium)
Preparation of 7.5% (m/v) sodium carbonate solution [0.71 M sodium carbonate
solution]. Weigh 7.5 g of sodium carbonate anhydrous into a 100 mL volumetric flask and
make up to the mark with distilled water.
Preparation of 2.5 mg/mL gallic acid stock solution. Weigh 0.250 g of gallic acid into a
100 mL volumetric flask and make up to the mark with distilled water.
Preparation of gallic acid standard solutions (in a range from 0.25 mg/mL to 1.25 mg/mL).
Five gallic acid standard solutions (Std. sol. I-0.25 mg/mL, Std. sol. II-0.50 mg/mL, Std.
sol. III-0.75 mg/mL, Std. sol. IV-1.00 mg/mL, and Std. sol. V-1.25 mg/mL) are prepared in
50 mL volumetric flasks using the gallic acid stock solution and distilled water in different
ratios. Prepare all gallic acid standard solutions in duplicate.
3.2.4. Apparatus and Accessories
• Analytical balance (ABJ-220-4NM; Kern & Sohn GmbH, Balingen, Germany)
• Vortex (Vortex V-1 Plus; Biosan Ltd., Riga, Latvia)
• Double beam UV-VIS spectrophotometer (UV-1900i; Shimadzu Scientific Instruments,
Inc., Columbia, MD, USA)
• Quartz cuvettes with lid, 1.4 mL, 10 mm path (Semi-Micro Cell 104-QS; Hellma
Analytics, Munich, Germany)
3.2.5. Working Procedure for Preparation of Calibration Curve
Preparation of Blank Sample
• Transfer 100 µL of methanol into a 16-mL glass bottle with a rubber stopper.
• Add 6 mL distilled water and 0.5 mL of 2 N Folin–Ciocalteu’s phenol reagent and mix
using the vortex.
• After 4 min, add 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled
water and vortex again.
• Keep the mixture in the dark, at room temperature, for 2 h.
• Turn on the spectrophotometer.
• Set the wavelength of the spectrophotometer to 750 nm.
• Transfer approximately 1.4 mL of the blank sample into each quartz cuvette.
• Insert the quartz cuvettes into the spectrophotometer.
• Press the “autozero” key.
Measurement of Standards (see Figure 1) for Calibration Curve Preparation
Figure 1. Visualization of standard solutions.
• Transfer 100 µL of the gallic acid standard solution into a 16-mL glass bottle with a
rubber stopper.
• Add 6 mL distilled water and 0.5 mL of 2 N Folin–Ciocalteu’s phenol reagent and mix
using the vortex.Molecules 2022, 27, 1345 8 of 13
• After 4 min, add 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled
water and vortex again.
• Keep the mixture in the dark, at room temperature, for 2 h.
• Transfer approximately 1.4 mL of the standard into the quartz cuvette from the front rack.
• Read and record the absorbance value of the standard (see Table 4).
Table 4. Worksheet for recording values of absorbances corresponding standards.
Standard Repetition Absorbance Value at 750 nm
1 0.190
Std. 1
2 0.187
1 0.405
Std. 2
2 0.392
1 0.597
Std. 3
2 0.613
1 0.820
Std. 4
2 0.828
1 1.058
Std. 5
2 1.081
• Repeat all above-mentioned steps for each standard.
• Generate the gallic acid calibration curve in Microsoft Excel by plotting values of
standards’ absorbances (Oy) vs their concentrations (Ox) (see Figure 2).
Figure 2. Gallic acid calibration curve for TPC evaluation.
3.2.6. Working Procedure for Measurement of TPC in Essential Oil
Preparation of Blank Sample
• Transfer 100 µL of methanol into a 16-mL glass bottle with a rubber stopper.
• Add 6 mL distilled water and 0.5 mL of 2 N Folin-Ciocalteu’s phenol reagent and mix
using the vortex.
• After 4 min, add 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled
water and vortex again.Molecules 2022, 27, 1345 9 of 13
• Keep the mixture in the dark, at room temperature, for 2 h.
• Turn on the spectrophotometer.
• Set the wavelength of the spectrophotometer to 750 nm.
• Transfer approximately 1.4 mL of the blank sample into each quartz cuvette.
• Insert the quartz cuvettes into the spectrophotometer.
• Press the “autozero” key.
Measurement of Test Sample
• Transfer 100 µL of the prepared test sample [thyme essential oil diluted in methanol
(20847.320P, VWR Chemicals, Fontenay-sous-Bois, France)-see Table 5] into a 16-mL
glass bottle with a rubber stopper.
Table 5. Preparation of thyme essential oil dilutions.
Dilution of TEO TEO [µL] Methanol [mL]
1:25 50 1.25
1:50 50 2.50
1:75 50 3.75
1:100 50 5.00
1:125 50 6.25
1:150 50 7.50
1:175 50 8.75
1:200 50 10.00
1:225 50 11.25
1:250 50 12.50
1:275 50 13.75
1:300 50 15.00
1:325 40 13.00
1:350 40 14.00
1:375 40 15.00
1:400 30 12.00
1:425 30 12.75
1:450 30 13.50
1:475 30 14.25
1:500 30 15.00
TEO—thyme essential oil.
• Add 6 mL distilled water and 0.5 mL of 2 N Folin–Ciocalteu’s phenol reagent and mix
using the vortex.
• After 4 min, add 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled
water and vortex again.
• Keep the mixture in the dark, at room temperature, for 2 h.
• Transfer approximately 1.4 mL of the test sample into the quartz cuvette from the front rack.
• Read and record the absorbance value of the test sample.
• Calculate TPC using the equation from Section 3.2.4 and record the value.
# Calculate the mean of the three values using the AVERAGE function in Excel.
# Calculate the standard deviation of the three values using the STDEV function
in Excel.
# Calculate the coefficient of variation (CV) by multiplying the ratio of standard
deviation to mean by 100.
• Report results as mean ± standard deviation of the three measurements.
3.2.7. Calculation and Expression of Results
Calculate the total phenolic content (TPC), expressed as mg gallic acid equivalent
(GAE)/100 µL essential oil, using the following Equation (4):
Abs. − b
m ×d
TPC = (4)
10Molecules 2022, 27, 1345 10 of 13
where Abs. is the absorbance value at 750 nm, b is the y-intercept of the linear equation, m
is the slope of the regression line, and d is the test sample dilution factor.
3.3. Identification and Quantification of Phenolic Compounds in Thyme Essential Oil by
HPLC-DAD-ESI-MS
3.3.1. Preparation of Thyme Essential Oil Methanolic Extract
It was carried out using the protocol described by Ricciutelli et al. (2017) [36], with
minor modifications. An aliquot (3 mL; ~2.83 g) of thyme essential oil was transferred
into a 15-mL Falcon centrifuge tube; 6 mL of n-hexane was added and vortexed (V-1 Plus
vortex; Biosan Ltd., Riga, Latvia) for 30 s, then 5 mL of methanol/water (3:2, v/v) and
sonicated for 15 min (Sonorex RK 100 H ultrasonic bath; Bandelin electronic GmbH &
Co. KG, Berlin, Germany). The mixture was then centrifuged at 867× g (3,000 rpm) for
10 min (EBA 20 centrifuge; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), and
the lower phase was collected (methanol-aqueous phase). Next, 3 mL of n-hexane was
added to the methanol-aqueous extract and vortexing, sonication, centrifugation, and phase
separation steps were repeated until a clear extract was obtained (three times). Finally,
the solvent was removed by evaporation at 40 ◦ C (Hei-VAP Expert rotary evaporator;
Heidolph Instruments GmbH & Co. KG, Schwabach, Germany), and the dry extract was
resuspended in 1 mL of methanol. The obtained methanolic extract was filtered through a
polyamide syringe filter (0.45 µm pore size, 25 mm diameter) then stored at −18 ◦ C until
chromatographic analysis.
Polyamide syringe filters (Chromafil Xtra PA 45/25; Macherey-Nagel GmbH & Co.
KG, Düren, Germany), n-hexane (Sigma-Aldrich Co., Saint Louis, MO, USA), and methanol,
LC-MS grade (Supelco, Inc., Bellefonte, PA, USA) were used as materials and reagents for
the extraction of polyphenolic compounds from thyme essential oil.
3.3.2. HPLC-DAD-ESI-MS Analysis of Thyme Essential Oil Methanolic Extract
It was performed using the method published by Fogarasi et al. (2021) [30]. Separation,
identification, and quantification of individual phenolic compounds were performed on
a liquid chromatography system (1200 HPLC; Agilent Technologies Inc., Palo Alto, CA,
USA). It contained a photodiode array (PDA) detector (G1315B), a single-quadrupole
mass spectrometer (MS) (G6110) equipped with an electrospray ionization (ESI) source
(G1948B). The system also included a quaternary pump (G1311A), a degasser (G1322A), an
autosampler (G1329A), a thermostatted column compartment (G1316A), a Kinetex XB-C18
column (150 mm L × 4.6 mm ID × 5 µm particle size; Phenomenex, Torrance, CA, USA), and
the ChemStation software (Rev B.04.02 SP1; Agilent Technologies Inc., Palo Alto, CA, USA).
Twenty microliters of thyme essential oil extract were injected into the HPLC system for
analysis. In the elution process, two mobile phases were employed: solution A containing
0.1% acetic acid in ultrapure water and solution B containing 0.1% acetic acid in acetonitrile.
A multi-step gradient elution model was used with the following settings: 5% B (0–2 min),
from 5 to 40% B (2–18 min), from 40 to 90% B (18–20 min), 90% B (20–24 min), from 90 to
5% B (24–25 min), 5% B (25–30 min). The flow rate was programmed to 0.5 mL/min and
the column oven temperature to 25 ◦ C. The PDA was set to scan from 200 to 600 nm. The
chromatograms were acquired at 280; data acquisition was performed for 30 min.
Mass spectra were recorded using the ESI source set in positive-ion mode and the MS
in full scan mode (in an m/z range of 120−1200). Nitrogen was used as a drying gas. Other
settings for the ESI source were as follows: drying gas temperature 350 ◦ C, drying gas flow rate
7 L/min, nebulizer pressure 35 psi, capillary voltage 3000 V, fragmentor voltage 100 eV.
Phenolic compounds were tentatively identified by comparing their retention times,
UV-vis spectra, and mass spectra to those of the standards analyzed under the same
conditions and data available in the literature [37].
Polyamide syringe filters (Chromafil Xtra PA 45/25; Macherey-Nagel GmbH & Co.
KG, Düren, Germany), glacial acetic acid, LC-MS grade (Supelco Inc., Bellefonte, PA, USA),
ultrapure water (Supelco Inc., Bellefonte, PA, USA), acetonitrile, LC-MS grade (SupelcoMolecules 2022, 27, 1345 11 of 13
Inc., Bellefonte, PA, USA), chlorogenic acid (Phytolab GmbH & Co. KG, Vestenbergsgreuth,
Germany), gallic acid (Phytolab GmbH & Co. KG, Vestenbergsgreuth, Germany), luteolin
(Phytolab GmbH & Co. KG, Vestenbergsgreuth, Germany), methanol, LC-MS grade (Su-
pelco Inc., Bellefonte, PA, USA), and nitrogen (SIAD România s.r.l., Bucharest, Romania)
were used as materials and reagents for the quantification of polyphenolic compounds by
HPLC-DAD-ESI-MS.
Standards of gallic acid, chlorogenic acid, and luteolin were prepared with methanol as
solvent. Hydroxybenzoic acids, terpenes, and stilbenes were quantified using a five-point
analytical curve of gallic acid (5−100 µg/mL; r2 = 0.9978); hydroxycinnamic acids using a
five-point analytical curve of chlorogenic acid (10−50 µg/mL; r2 = 0.9937); flavones and
flavanones using a five-point analytical curve of luteolin (0−100 µg/mL; r2 = 0.9972). The
limit of detection [LOD = 3.3 × (Sy-the standard deviation of the response/S-the slope of
the calibration curve)] was 0.35 µg/mL, and the limit of quantification [LOQ = 10 × (Sy/S)]
was 1.05 µg/mL in the case of gallic acid; 0.41 µg/mL LOD and 1.64 µg/mL LOQ in case
of chlorogenic acid; 0.21 µg/mL LOD and 0.84 µg/mL LOQ in case of luteolin. Samples
were tested in triplicate. Results were expressed in µg/mL essential oil.
3.3.3. Statistical Analysis
For data analysis, Minitab statistical software (version 19.1.1; LEAD Technologies, Inc.,
Charlotte, NC, USA) was used. The difference between values of dilutions was determined
using one-way ANOVA. Post-hoc pairwise comparisons were performed with Tukey’s test
at a 95% confidence level (p < 0.05).
4. Conclusions
This paper provides a laboratory procedure for spectrophotometric evaluation of TPC
in thyme essential oil, containing instructions for conducting a calibration experiment
and explanations on how to evaluate results. All analytical parameters analyzed showed
adequate results after implementing the conditions for colorimetric assessment of phenolic
compounds by the Folin-Ciocalteu’s phenol reagent. Furthermore, given the details pro-
vided by this procedure, researchers can easily replicate or use then in their experimental
laboratory investigations. Moreover, they can routinely use the proposed protocol to assess
TPC in any methanolic extract.
Author Contributions: Conceptualization, C.A.S. and A.M.J.; methodology, C.A.S. and D.M.; anal-
ysis, D.M., M.-I.S., M.F. and F.R.; writing—original draft preparation, D.M.; writing—review and
editing, C.A.S.; visualization, V.M.; supervision, C.A.S.; project administration, V.M. and M.F.;
funding acquisition, V.M. and M.F. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by two grants of the Ministry of Research, Innovation and
Digitization, CNCS-UEFISCDI, project number PN-III-P2-2.1-PED-2019-5346 and project number
PN-III-P1-1.1-PD-2019-0475, within PNCDI III. The publication was supported by funds from the
National Research Development Projects to finance excellence (PFE)-14/2022-2024 granted by the
Romanian Ministry of Research and Innovation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We are grateful for the administrative and financial support offered by the
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.Molecules 2022, 27, 1345 12 of 13
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